For propagation of plants, various methods such as seeding, cuttage, division or suckering, tuber, bulb, and tuberous root have been employed. However, where the clonal propagation is sought, among tissue culture methods, in particular use of a propagation method utilizing a somatic embryo has been demanded, and in some occasions, its practical use has been examined (Pramod et al., Scale-up and automation in plant propagation: 76-93, 1991, Academic Press, Inc.). In conventional methods for inducing a somatic embryo, however, because the induced somatic embryo population frequently contains a high rate of somatic embryo masses (Mamiya and Sakamoto, J. of Plant Physiol., 159: 553-556, 2002; Chi et al., Biotechnology and Bioengineering 50: 65-7; and Chi et al., Biotechnology and Bioengineering 50: 65-72, 1996), a variety of means to recover a single somatic embryo which is more suitable for mass propagation have been proposed. However, the somatic embryo mass which occupies a high rate of somatic embryo population has not been actively utilized except plant species capable of permitting a multiple shoots body, resulting in a big problem in improving propagation efficiency. Even for plants in which an example of inducing a somatic embryo is known, the above situation is one of big factors responsible for a reason that the clonal propagation using the somatic embryo cannot be realized.
As a method for recovering a single somatic embryo from a somatic embryo population, a technique for screening a somatic embryo having a certain size or less and naturally passing through the openings of a mesh using the mesh without physical treatment (Nadel et al., Plant Cell, Tissue and Organ Culture 20: 119-124, 1990) and a technique of screening utilizing image analysis (Padmanabhan et al., Plant Cell Reports 17: 681-684 and Harrell et al., Acta Horticulturae 319: 595-600, 1992) have been proposed. However, these techniques primarily employ a method in which an existing single somatic embryo is simply screened from a somatic embryo population. As described above, the method for recovering a single somatic embryo from the somatic embryo mass which occupies a high rate of somatic embryo population is not contemplated. In the meantime, by modifying the composition of a medium for inducing a somatic embryo, an attempt to enhance an efficiency of inducing a single somatic embryo has been made in order to yield a certain outcome (Mamiya and Sakamoto, J. of Plant Physiol., 159: 553-556, 2002 and Dai, In Vitro Cell. Dev. Biol.-Plant 40: 376-383, July/August 2004). However, a procedure actively utilizing a somatic embryo mass, for which a universal technique common to many plants is sought, has not been reported so far, wherein the procedure comprises efficiently inducing a single somatic embryo.
A straining method utilizing a mesh has been applied to a callus (a kind of cultured cell) in some cases, and has achieved a certain effect such as an improvement in efficiency of somatic embryogenesis (Noguchi, Bulletin of the Tokyo Agricultural Experiment Station 27: 1-8, 1997 (Japan)). A cultured cell such as a callus is different from a somatic embryo, and constitutes an undifferentiated tissue. Since the tissue unit is small and the binding affinity between the tissues is weak, the damage due to division is small. However, the somatic embryo is a definitely differentiated tissue, and a method for physically dividing a somatic embryo mass is easily predicted to have extremely large damage to the tissue. Hence, there is no example that this division method has been examined.
As a method of utilizing the somatic embryo mass, known is a method comprising subjecting a somatic embryo mass to a germination process utilizing a solid medium, etc.; and dividing the somatic embryo mass into individual seedlings after the germination. However, such division operation would consume labor and time, resulting in high costs. Accordingly, the applicable plant species remains limited. In addition, although separation of the somatic embryos one by one from the somatic embryo mass may be allowed in some conditions, this technique would produce more troublesome operation compared with the case of dividing a germinating body, so that the technique is not a practical method.